Conversion of 7-dehydrocholesterol to cholesterol is the last reaction in the cholesterol biosynthesis pathway catalyzed by the microsomal enzyme 7-dehydrocholesterol-delta 7 reductase (EC 1.3.1.21). Inhibiting the last step in cholesterol biosynthesis profoundly reduces tissue and plasma cholesterol concentrations and accumulates precursors that substantially slow hepatoma growth. Inhibiting late cholesterol synthesis also hinders the growth of rapidly enlarging malignant tumors (Xu, G. et al. (1996) Hepatology 24:440-445). Analyses of the cDNA encoding the human delta 7 sterol reductase shows that this enzyme is a membrane-bound protein containing 6 to 9 putative transmembrane segments and is structurally related to plant and yeast sterol reductases. The delta 7 sterol reductase is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human delta 7 reductase cDNA remove the C7-8 double bond in 7-dehydrocholesterol in a NADPH-dependent manner (Moebius, F. F. et al. (1998) Proc. Natl. Acad. Sci. USA 95:1899-1902).
A microsomal preparation from seedlings of Zea mays catalyzed the NADPH-dependent reduction of the delta 7 bond of delta 5,7 cholestadienol giving the first in vitro evidence of the intermediacy of delta 5,7 sterols in plant sterol biosynthesis. In vitro inhibition of the plant delta 5,7 sterol delta 7 reductase by ammonium ion-containing fungicides was consistent with the previously proposed cationic mechanism involved in this reduction reaction (Taton, M. and Rahier, A. (1991) Biochem. Biophys. Res. Commun. 181:465-473). The NADPH-sterol delta-7 reductase from Arabidopsis thaliana has been cloned. The corresponding protein has significant sequence similarity with yeast delta 14 and delta 24 reductases and with human lamin B receptor. This protein is capable of efficiently reducing in vivo delta-5,7-ergosta- and cholesta-sterols, regardless of the structural variations on the side chain. The delta 7 reductase activity is preferentially associated with the endoplasmic reticulum membrane and uses NADPH as the reducing agent (Lecain, E. et al. (1996) J. Biol. Chem. 271:10866-10873).
Regulation of sterol biosynthesis in the terminal portion of the pathway represents an efficient mechanism by which the cell can control the production of sterol without disturbing the production of other essential mevalonate pathway products. Expression of ERG3, the gene encoding sterol C-5 desaturase, is increased in response to a mutation in the major isoform of HMG-CoA reductase which catalyzes the rate-limiting step of sterol biosynthesis. Mutations in non-auxotropic ergosterol biosynthetic genes downstream of squalene production result in an up-regulation of ERG3 expression. Absence of sterol esterification leads to a decrease in total intracellular sterol and ERG3 is a target of this negative regulation (Arthington-Skaggs, B. A. et al. (1996) FEBS Lett. 392:161-165). ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol delta 5 desaturase that introduces the C5=6 unsaturation in ergosterol biosynthesis. Inactivated mutants of ERG3 fail to grow without added levels of delta 5 sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol (Smith, S. J. and Parks, L. W. (1993) Yeast 9:1177-1187). A construct containing the promoter for the ERG3 gene fused to the bacterial lacZ reporter gene was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as 9-fold in erg-mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifingal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae (Smith, S. J. et al. (1996) Mol. Cell Biol. 16:5427-5432). A human cDNA clone homologous to fungal ERG3, a gene encoding sterol C-5 desaturase has been isolated. This gene is expressed in all normal human tissues examined (Matusushima, M. et al. (1996) Cytogent. Cell Genet. 74:252-254).